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Abstract

Background

Acute myeloid leukemia (AML) and other aggressive refractory hematological malignancies
unresponsive to upfront therapy remain difficult conditions to treat. Often, the focus
of therapy is centered on achieving complete remission of disease in order to proceed
with a consolidative stem cell transplant. At issue with this paradigm is the multitude
of patients who are unable to achieve complete remission with standard chemotherapeutic
options. A major benefit of transplantation is the graft versus tumor effect that
follows successful engraftment. However, with this graft versus tumor effect comes
the risk of graft versus host disease. Therefore, alternative treatment options that
utilize immunotherapy while minimizing toxicity are warranted. Herein, we propose
a novel treatment protocol in which haploidentical peripheral blood stem cells are
infused into patients with refractory hematological malignancies. The end goal of
cellular therapy is not engraftment but instead is the purposeful rejection of donor
cells so as to elicit a potent immune reaction that appears to break host tumor tolerance.

Methods/design

The trial is a FDA and institutional Rhode Island Hospital/The Miriam Hospital IRB
approved Phase I/II study to determine the efficacy and safety of haploidentical peripheral
blood cell infusions into patients with refractory hematological malignancies. The
primary objective is the overall response rate while secondary objectives will assess
the degree and duration of response as well as safety considerations. Patients with
refractory acute leukemias and aggressive lymphomas over the age of 18 are eligible.
Donors will be selected amongst family members. Full HLA typing of patients and donors
will occur as will chimerism assessments. 1-2x108 CD3+ cells/kilogram will be infused on Day 0 without preconditioning. Patients will
be monitored for their response to therapy, in particular for the development of a
cytokine release syndrome (CRS) that has been previously described. Blood samples
will be taken at the onset, during, and following the cessation of CRS so as to study
effector cells, cytokine/chemokine release patterns, and extracellular vesicle populations.
Initially, six patients will be enrolled on study to determine safety. Provided the
treatment is deemed safe, a total of 25 patients will be enrolled to determine efficacy.

Discussion

Cellular Immunotherapy for Refractory Hematological Malignancies provides a novel
treatment for patients with relapsed/refractory acute leukemia or aggressive lymphoma.
We believe this therapy offers the immunological benefit of bone marrow transplantation
without the deleterious effects of myeloablative conditioning regimens and minus the
risk of GVHD. Laboratory correlative studies will be performed in conjunction with
the clinical trial to determine the underlying mechanism of action. This provides
a true bench to bedside approach that should serve to further enrich knowledge of
host tumor tolerance and mechanisms by which this may be overcome.

Trial registration

Keywords:

Background

Despite recent improvements in the care of patients with acute myeloid leukemia (AML),
the overall prognosis remains dismal with a 5-year relative survival rate of slightly
over 20% [1]. Typically, AML patients eligible for curative therapy are treated with high dose
chemotherapy involving an anthracycline. If remission is achieved, the majority of
patients undergo allogeneic stem cell transplantation (allo-SCT) for definitive curative
treatment if, based on age and other medical co-morbidities, they are deemed clinically
eligible [2]. Much of the therapeutic effect of stem cell transplantation arises from a graft
versus leukemia (GVL) response. Unfortunately, GVL is associated with potential complications
such as treatment-related morbidity/mortality from conditioning chemotherapy regimens
as well as graft-versus-host disease (GVHD). Furthermore, even after complete response
and transplantation there is a 30% probability of disease relapse, and patients who
experience a relapse have a 2-year overall survival (OS) of 10-14% [3].

Other hematological malignancies such as acute lymphocytic leukemia (ALL) employ allo-SCT
as upfront therapy in poor prognostic settings such as Philadelphia chromosome positive
ALL while others, like aggressive non-Hodgkin lymphoma (NHL), utilize allo-SCT in
cases of relapsed disease. As is the case in AML, patients with ALL and NHL requiring
allo-SCT have poor 5 year OS of 30-38% [4-6] and 24-27% [7,8], respectively. Similar to AML, allo-SCT of relapsed ALL and NHL is limited by treatment
related morbidity/mortality [7-9]. Hence, additional therapeutic modalities for acute leukemia and aggressive lymphomas
are needed. Herein, we propose an alternative mechanism for cell-directed immunotherapy
in relapsed and refractory hematological malignancies that does not require high-dose
chemotherapy for conditioning. In our protocol, termed cellular immunotherapy, the
goal of cell infusion is not engraftment but instead is the purposeful rejection of
donor cells so as to elicit a complex cytokine storm that, we believe, breaks host
tumor tolerance.

Laboratory origins of cellular immune therapy

The origins of cellular immunotherapy stem from early work describing engraftment
without myeloablation. Based on the previous work of others, we initially studied
the capacity of murine marrow cells to engraft into non-irradiated mice [10]. From the experimental data obtained, we concluded that donor cell engraftment is
not only possible without myeloablation but is also quantitative in nature by virtue
of competition between host and donor cells [11]. Greater numbers of donor cells infused results in higher levels of engraftment.
However, in the clinical setting non-myeloablative engraftment is not possible due
to the high number of donor cells that would be required. Therefore, we next examined
levels of syngeneic murine engraftment with minimal myeloablation. Utilizing small
doses of radiation (50-100 cGy) we demonstrated significant engraftment [12]. The thought behind efficacy in minimal myeloablation was that small radiation doses
were stem cell toxic but not myelotoxic. Initial attempts by our group to translate
these findings to the allogeneic setting were unsuccessful secondary to immune barriers.
Eventually, however, durable non-myeloablative engraftment was possible through anti-CD40
ligand antibody co-stimulatory blockade in the setting of multiple stem cell infusions
[13].

Clinical origins of cellular immune therapy

Infusion of HLA identical pheresed cells

The above murine studies became the foundation of a clinical approach for patients
with refractory leukemia and lymphoma. Initially, we infused graded doses of human
leukocyte antigen (HLA) identical allogeneic T-cells into eleven minimally myeloablated
(100 cGy total body irradiation) patients with refractory hematological malignancies
in an attempt to achieve durable engraftment. Nine patients achieved mixed or complete
chimerism. Four patients attained a complete response (CR), two of which were of long
duration. CR was achieved in the single patient treated for refractory AML, in two
patients with NHL and in one patient with multiple myeloma (MM). Five patients developed
significant acute GVHD, resulting in one death. Of the patients achieving a CR, three
developed 100% chimerism. The fourth patient in CR had only a transient 5% chimerism
for one week but interestingly had a sustained CR of over 42 months. Another patient
with chronic lymphocytic leukemia (CLL) showed a 75% reduction in lymphadenopathy
despite no evidence for chimerism thereby suggesting that the cellular infusion may
have activated the patient’s own immune system against their hematologic malignancy.
In the end, we determined the infusion of 1 × 108 T-cells per kg along with a median of 5 × 104 CD34+ cells per kg from non-mobilized HLA identical blood was safe, effective, and
warranted further clinical study [14].

Infusion of HLA haploidentical pheresed cells

In order to increase the number of patients eligible for cellular infusion, we next
evaluated the infusion of haploidentical peripheral blood cells into 26 patients with
refractory hematologic malignancies [15]. Granuloctye colony stimulating factor (G-CSF) mobilization was used for collection
of peripheral blood cells. Following 100 cGy of total body irradiation (TBI), patients
were infused with escalating levels of CD3+ cells. The study recruited thirteen patients
with AML, six with NHL, five with MM, one with ALL and one with chronic myeloid leukemia
(CML). No responses were seen in the eight patients treated with 1 × 106 or 1 × 107 CD3+ cells per kg. At higher CD3+ dose levels of 1–2 × 108 per kg, objective responses were seen in 14 out of 18 patients. Two of six patients
with NHL remained free of disease at 76 months and 82 months, respectively, while
two additional NHL patients obtained partial responses (PR). There were 3 durable
CR lasting 8, 11 and 31 months, respectively, and 7 transient responses in 13 patients
with AML (Table 1). Remarkable, all responses occurred outside of donor chimerism. Serial bone marrow
biopsies performed in several patients showed evidence of large tumor reduction and
early resumption of normal hematopoiesis.

Toxicities included well-tolerated myelotoxicity. An immediate post-infusion immunologic
syndrome, which we termed “haploimmunostorm”, was observed. This was universally characterized
by fever and variably by skin rash, diarrhea, liver dysfunction, effusions, respiratory
distress and edema. These signs and symptoms were variable between patients. It only
occurred in patients infused with at least 1 × 108 CD3+ cells per kg, began 14 hours after cell infusion, and remitted rapidly with
methylprednisolone, 2 mg/kg/day within 6–8 hours of the onset of the haploimmunostorm.
Typically, we allowed this syndrome to persist for at least 48 hours as it was hypothesized
that this might be a component of the therapeutic response.

Donor chimerism, as determined by short tandem repeat testing in multiplex battery
with 1-5% sensitivity, was not seen in most patients. Only two patients developed
donor chimerism and both patients died; one patient clearly died of GVHD and the other
possibly from GVHD. Hence, we were able to document complete responses, some of which
were durable, with cell infusions in the absence of demonstrable engraftment.

Evaluation of serum cytokine levels during the haploimmunostorm revealed elevations
of multiple cytokines including interferon γ which in the murine model has been shown
to play a role in the host versus tumor response [16,17]. The pattern of cytokine elevations was distinct from the patterns seen with the
engraftment syndrome and GVHD. These elevations were felt to be related to the haploimmunostorm
manifestations.

Cytokine release syndrome; other studies

Similar to the description of the haploimmunostorm phenomenon is a cytokine release
syndrome (CRS) that has been observed in the chimeric antigen receptor (CAR) modified
T-cells studied in CLL patients by the University of Pennsylvania group [18]. In their work, the cytokines IFN γ and IL-6 are elevated in patients whose CLL responds
to infusion of modified CAR T-cells while those patients who exhibit no CRS show no
response to this therapy. These initial findings in CLL have been reproduced in patients
with ALL [19]. The clinical findings of hypotension and hypoxia are also similar to our experiences
with haploimmunostorm, as is the use of steroids to treat these symptoms. More recently,
they have shown blockade of IL-6 with the monoclonal antibody tociluzimab is able
to mitigate the side effects of cytokine release syndrome without dampening the anti-tumor
activity [20].

Nonengraftment haploidentical cellular therapy; other studies

The clinical efficacy of cellular therapy has been replicated by Guo and colleagues
in a study of patients ≥ 60 with AML [21]. Here, they randomized patients to either receive chemotherapy alone or chemotherapy
with haploidentical G-CSF mobilized peripheral blood stem cells (PBSC). In the chemotherapy
alone group, the CR rate was 43% while the CR rate in the chemotherapy and PBSC group
was 80%. Furthermore, the 2-year progression free survival (PFS) with chemotherapy
alone was 10% in contrast to a 2-year PFS of 39% in the chemotherapy and PBSC cohort.
These results show clear activity for cellular therapy in patient population with
a notoriously dismal prognosis. A subsequent study performed by Guo et al. infused
HLA-mismatched donor G-CSF mobilized PBSC following 3 cycles of cytarabine for AML
patients who attained CR after induction chemotherapy. In total, 101 low- and intermediate-risk
AML patients were included in this study. In the low-risk group, the 6-year leukemia
free survival (LFS) rate was 84% while the OS was 89%. In the intermediate-risk group
the LFS was 59% and the OS was 65% [22]. These are remarkable responses for patients with AML in whom the average 5-year
OS is 55% and 24% in similar risk groups [23]. Interestingly, this same group reported a benefit of donor cell infusions with chemotherapy
for the treatment of myelodsyplastic syndrome compared to chemotherapy alone [24].

Study rationale

The infusion of 1–2 × 108 CD3+ haploidentical cells per kg into minimally irradiated patients with refractory
lymphoma or leukemia results in dramatic and sometimes durable responses in the absence
of engraftment [15,21,22]. In this setting, the patients developed a unique CRS characterized by fever, diarrhea,
liver function abnormalities, skin rash and pulmonary symptoms. This suggests the
activity in haploidentical blood cell infusion is likely due to activation of the
recipient’s immune system against leukemia/lymphoma and not due to graft versus tumor.
This response may be related to CRS development as described by our group [15] and others [18,20].

In order to increase the host immune response to haploidentical cellular infusion,
no pre-infusion irradiation or chemotherapy will be administered. Furthermore, as
G-CSF would be administered to healthy volunteers the unclear benefit of the addition
of this cytokine is offset by the potential side effects such as headache, fever,
and bone pain. Moreover, G-CSF mobilization serves to shift the response from a TH1 to TH2 through the increased production of T-regulatory cells thereby potential decreasing
the immune response [25]. Therefore, in this study, haploidentical cells will be collected directly from the
donor and infused into the patient. The end goal is purposeful rejection of donor
cells by the host immune system which, we postulate, results in breakage of host tumor
tolerance. The underlying mechanism behind this phenomenon has not been fully elucidated
but is thought to involve a complex interaction between interferon γ, CD8+ T-cells,
NK cells, and antigen presenting cells. Laboratory correlative studies to determine
the mechanism of action will be conducted alongside the clinical trial.

Methods/design

The current trial is a single-arm phase I/II non-randomized study designed to evaluate
the safety and efficacy of haploidentical cellular infusion in patients with refractory
acute leukemia and aggressive lymphoma. Additional laboratory correlative studies
will be performed in conjunction with the clinical trial to determine an underlying
mechanism of action.

Primary and secondary objectives

The primary objective is to assess the overall response rate of cellular immune therapy
with HLA-haploidentical peripheral blood pheresed cells in patients with relapsed/refractory
hematological malignancies.

Secondary objectives will more accurately describe the clinical effect by assessing
the time-to-progression, PFS and OS for patients with relapsed/refractory hematologic
malignances following HLA-haploidentical cellular therapy. An additional secondary
objective is to evaluate the rate of dose-limiting toxicities of HLA-haploidentical
peripheral blood pheresed cellular infusions.

Exploratory objectives

These objectives will serve to decipher the underlying process by which cellular therapy
results in clinical response. We will evaluate in vitro mixed lymphocyte assays of donor and recipient cells to determine if in vitro stimulation and cytolytic activity corresponds to clinical efficacy. Furthermore,
samples will be taken prior to, during, and after the onset of the cytokine release
syndrome in order to determine cytokine release profiles, effector cell populations,
and extracellular vesicle release.

Study design

All patients over the age of 18 with relapsed aggressive lymphoma or acute myeloid/lymphoid
leukemia with at least one prior therapy and no curative options are eligible (Table 2). HLA-haploidentical donors over the age of 18 whom are healthy and meet criteria
of blood donation are eligible (Table 3).

Selected donors will undergo leukapheresis. The final product will be analyzed for
CD3+ and CD34+ content via flow cytometry. The product will be administered unprocessed
on day 0 (same day as leukapheresis) with 1–2 × 108 CD3+ cells per kg irrespective of the number of CD34+. No specific viral or bacterial
prophylaxis is required. The infusion of HLA-haploidentical peripheral blood cells
must be initiated within 8 hours of product collection and completed within 24 hours.
Acetaminophen 650 mg PO and diphenhydramine 50 mg IV will be administered 30 minutes
prior to haploidentical infusion. During the infusion, patient will be monitored for
blood pressure, temperature and oxygen saturation. This monitoring will continue for
2 hours after infusion at the following time points; every 15 minutes the first hour
post infusion and every 30 minutes the second hour post infusion.

The infusion will be stopped should the patient develop grade 3 or 4 infusion-related
reactions. For grade 2 infusion-related reactions the following protocol will be followed:

1st occurrence of Grade 2 infusion related reaction

1. Infusion placed on hold

2. Acetaminophen 650 mg PO × 1

3. Diphenhydramine 50 mg IV × 1

4. Ranitidine 50 mg IV ×1

5. Infusion restarted following improvement in symptoms to Grade 1 or less

2nd occurrence of Grade 2 infusion related reaction

1. Infusion placed on hold

2. Methylprednisolone 100 mg IV × 1

3. Infusion restarted following improvement in symptoms to Grade 1 or less

Toxicities/Safety

Patients will be monitored for cell infusion syndrome and cytokine release syndrome.
Patients will be given methylprednisolone for Grade 3 or 4 Toxicities based on NCI
CTC version 4.0 criteria. Furthermore, percent chimerism will be assessed using short
tandem repeats 2 days post cellular infusion, 14 post cellular infusion, and every
14 days thereafter as long as chimerism persists (Figure 1). Lack of chimerism will obviate the risk of GVHD. After 6 patients have been followed
for a minimum of 2 months after cellular infusion, the Brown University Oncology Research
Group (BrUOG) will review safety data prior to reopening accrual. Data from the safety
review will be shared with the FDA.

Adverse events will be scored according to the NCI CTCAE version 4 criteria. Dose
limiting toxicity (DLT) will be defined as any of the following treatment related
events:

• Grade 3 graft versus host disease lasting > 7 days

• Grade 4 graft versus host disease of any duration

• Grade 3 infusion related symptoms lasting > 7 days

• Grade 4 infusion related symptoms of any duration

In the safety run-in for the first 6 patients, if 2 or less of 6 patients have a dose
limiting toxicity then accrual will be allowed to extend to a total of 25 patients.
If 3 or more of 6 patients have a DLT, or there is one grade 5 treatment-related adverse
events, protocol accrual will be suspended. If this circumstance occurs, the BrUOG
data safety monitoring group will review the adverse event data and make appropriate
recommendations to the BrUOG scientific advisory board and the FDA about the study.

The safety evaluation of all 25 evaluable patients is a major objective of this study.
A 35% rate of treatment-related adverse events from cellular infusion will be considered
unacceptable.

Table 4.Response criteria for cellular immunotherapy in both leukemia and lymphoma [[26],[27]]

Statistical considerations

As noted above, the safety of cellular therapy administration in the first six patients
will be reviewed by the FDA and the BrUOG Data Safety Monitoring Board (DSMB). These
reviews will be shared with our institutional IRB. If the treatment appears safe,
as determined by the FDA and the BrUOG DSMB with institutional IRB agreement, the
protocol may be reopened to treat a total of 25 patients. A sample size of 25 patients
will differentiate between a 10% level of activity and a 30% level of activity. Specifically,
the hypothesis to be tested is:

H0: p< 0.1 versus H1: p> 0.3

A Simon two-stage design will be used in this study. The first 15 evaluable patients
will be assessed for activity. The trial will be terminated early if activity is observed
in 0 or 1 of these 15 patients, and it will be concluded that the true activity rate
is unlikely to be > 10%. If activity is demonstrated in at least 2 patients, accrual
will continue until a total of 25 evaluable patients are enrolled. If activity is
observed in 5 or fewer of 25 patients, the null hypothesis will be accepted and it
will be concluded that there is not sufficient activity to merit further investigation
of the regimen. Otherwise, it will be concluded that the treatment regimen has sufficient
activity to warrant further investigation.

The characteristics of this study design are as follows:

This design yields a type I error rate of <0.05 (α=0.03) and power of 80% when the
true response rate is 30%.

Overall survival, time to progression, and progression free survival will be determined
by the Kaplan Meier method (from the time of study enrollment).

Translational endpoints

Prospective studies for evaluation of donor and patient alloreactivity

Within our center we have created an in vitro study in which inactivated randomly selected mismatched donor cells are mixed with
CD3+ cells from leukemia patients. Stimulated CD3+ patient cells are then placed on
51Cr labeled leukemic blasts with cytolytic activity measured by 51Cr release. Preliminary results obtained thus far show cytolytic anti-leukemic activity
in approximately half of the stimulated patient CD3+ cells [28]. Because these results are about the same frequency as the responses to cellular
immunotherapy, it raises the question of whether this in vitro assay would be predictive of in vivo responses using the donor/patient combination to be tested. Further, if CD3+ proliferation
and cytolytic activity, as determined by in vitro assays, is donor dependant then it may be possible to identify an optimal donor.
These in-vitro studies will be done in a prospective manner by obtaining additional tubes of blood
from patient and donor at the day −28 time point (Figure 1). Blood will also be obtained from other individuals that could have been considered
as donors or from completely mismatched normal controls.

Identification of functional effector cells after cellular infusion

Blood samples will be taken frequently during the first few days after cellular infusion
(Figure 1). Blood will be centrifuged with the plasma collected from these samples and aliquots
frozen down for future analysis. PBMC will be isolated from the cell pellet using
Ficoll-Hypaque discontinuous centrifugation. An aliquot of PBMC will be frozen down
for killer-cell immunglobulin like receptor (KIR) expression using Luminex based typing
(Gen-Probe) [29]. PBMC analysis will include cell staining for the presence of various subpopulations
and their activation status. A panel of anti-HLA antibodies coupled with AlexaFluor
488 that has been shown to be useful for detecting microchimerism during pregnancy
in 90% of individuals tested has been made available [30,31]. These antibodies will be used to distinguish donor and recipient cells. In addition
to determining the percentage of donor cells circulating in the blood at these different
time points, these antibodies can be combined with antibody staining panels that define
different subpopulations (CD3, CD4, CD8, CD56, CD69), as well as cytokine and granzyme
production by these cells. If there are sufficient cells, it may be possible to use
antibody panel staining with anti-HLA antibodies to sort for donor and/or recipient
cells. Once collected, these populations could be tested directly for their ability
to lyse leukemic target cells or natural killer target cells such as K562. If there
are limited number of cells, PCR will be performed to determine the expression of
loci expressing effector molecules.

Discussion

Cellular Immunotherapy for Refractory Hematological Malignancies provides a novel
treatment for patients with relapsed and refractory acute leukemia or aggressive lymphoma.
We believe this therapy could offer the immunological benefit of bone marrow transplantation
(i.e. GVL) without the deleterious effects of myeloablative conditioning regimens
and the risk of GVHD. Although the exact mechanism of action behind the clinical efficacy
remains to be elucidated, we believe that the potential therapeutic benefit is too
great to ignore. Furthermore, laboratory correlative studies will be performed in
conjunction with the clinical trial. This provides a true bench-to-bedside approach
that should serve to further enrich knowledge of host tumor tolerance and mechanisms
by which this may be overcome.

The overriding goal of this phase I/II study is to generate clinical responses with
a treatment that is safe and well tolerated. The initial six patients enrolled will
be re-evaluated for safety considerations with potential alterations to the protocol
made in order to enhance both safety and efficacy. The purposeful rejection phenomenon
seen within our study could serve as a platform for additional trials of host-mediated
immunotherapy for other malignancies.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

JLR, HS, ESW, JJC, JNB, MIQ, CTY, and PJQ designed the protocol. LDF and MN developed
the in vitro assay. JLR, LDF, ESW, JJC, and PJQ wrote the manuscript. All authors reviewed and
approved the final draft of the manuscript.

Authors’ information

JLR was recently awarded a Leukemia and Lymphoma Society Special Fellow in Clinical
Research Grant for the above work.

Acknowledgements

This publication was made possible by Grant Number 8P20GM103468 from National Institutes
of Health/National Institute of General Medical Sciences and its contents are solely
the responsibility of the authors and do not necessarily represent the official views
of the NIH/NIGMS.

Sponsor/Funding

The study is sponsored by the Brown University Oncology Research Group, the Division
of Hematology/Oncology at Rhode Island Hospital and The Miriam Hospital, and the Center
for Biomedical Research Excellence at Rhode Island Hospital.

Study approval

The study is approved by the United States Food and Drug Administration and by the
Rhode Island Hospital Institutional Review Board.